Pradip Roy-Burman

Pten Dose Dictates Cancer Progression in the Prostate

Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Ptenhy/+ > Pten+/− > Ptenhy/− (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Ptenpc) mutants. In addition, we have generated and comparatively analyzed two distinct Ptenpc mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27Kip1, mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression.

Generation of a prostate epithelial cell-specific Cre transgenic mouse model for tissue-specific gene ablation

To facilitate the elucidation of the genetic events that may play an important role in the development or tumorigenesis of the prostate gland, we have generated a transgenic mouse line with prostate-specific expression of Cre recombinase. This line, named PB-Cre4, carries the Cre gene under the control of a composite promoter, ARR2PB which is a derivative of the rat prostate-specific probasin (PB) promoter. Based on RT-PCR detection of Cre mRNA in PB-Cre4 mice or Cre-mediated activation of LacZ activity in PB-Cre4/R26R double transgenic mice, it is conclusively demonstrated that Cre expression is post-natal and prostatic epithelium-specific. Although the Cre recombination is detected in all lobes of the mouse prostate, there is a significant difference in expression levels between the lobes, being highest in the lateral lobe, followed by the ventral, and then the dorsal and anterior lobes. Besides the prostate gland, no other tissues of the adult PB-Cre4 mice demonstrate significant Cre expression, except for a few scattered areas in the gonads and the stroma of the seminal vesicle. By crossing the PB-Cre4 animals with floxed RXRalpha allelic mice, we demonstrate that mice, whose conventional knockout of this gene is lethal in embryogenesis, could be propagated with selective inactivation of RXRalpha in the prostate. Taken together, the results show that the PB-Cre4 mice have high levels of Cre expression and a high penetrance in the prostatic epithelium. The PB-Cre4 mice will be a useful resource for genetic-based studies on prostate development and prostatic disease.

Synergy of p53 and Rb Deficiency in a Conditional Mouse Model for Metastatic Prostate Cancer

Pathways mediated by p53 and Rb are frequently altered in aggressive human cancers, including prostate carcinoma. To test directly the roles of p53 and Rb in prostate carcinogenesis, we have conditionally inactivated these genes in the prostate epithelium of the mouse. Inactivation of either p53 or Rb leads to prostatic intraepithelial neoplasia developing from the luminal epithelium by 600 days of age. In contrast, inactivation of both genes results in rapidly developing (median survival, 226 days) carcinomas showing both luminal epithelial and neuroendocrine differentiation. The resulting neoplasms are highly metastatic, resistant to androgen depletion from the early stage of development, and marked with multiple gene expression signatures commonly found in human prostate carcinomas. Interestingly, gains at 4qC3 and 4qD2.2 and loss at 14qA2-qD2 have been consistently found by comparative genomic hybridization. These loci contain such human cancer-related genes as Nfib, L-myc, and Nkx3.1, respectively. Our studies show a critical role for p53 and Rb deficiency in prostate carcinogenesis and identify likely secondary genetic alterations. The new genetically defined model should be particularly valuable for providing new molecular insights into the pathogenesis of human prostate cancer.

Pten null prostate tumorigenesis and AKT activation are blocked by targeted knockout of ER chaperone GRP78/BiP in prostate epithelium

GRP78/BiP has recently emerged as a novel biomarker for aggressive prostate cancer. Here, we report that homozygous deletion of Grp78 specifically in mouse prostate epithelium suppresses prostate tumorigenesis without affecting postnatal prostate development and growth. Mouse prostates with double conditional knockout of Grp78 and Pten exhibit normal histology and cytology, in contrast to the invasive adenocarcinoma in mouse prostates with Pten inactivation. AKT activation in Pten null prostate epithelium is inhibited by Grp78 homozygous deletion, corresponding with suppression of AKT phosphorylation by GRP78 knockdown in prostate cancer cell line. Thus, inactivation of GRP78 may represent a previously undescribed approach to stop prostate cancer and potentially other cancers resulting from the loss of PTEN tumor suppression and/or activation of the oncogenic AKT.

A Novel Association between the Human Heat Shock Transcription Factor 1 (HSF1) and Prostate Adenocarcinoma

A search for differentially expressed genes in a pair of nonmetastatic (PC-3) versus metastatic variant (PC-3M) human prostate carcinoma cell lines led to identification of the human heat shock factor (HSF1) as an overexpressed gene product in PC-3M cells. Analysis of primary prostate cancer specimens indicated that HSF1 is generally up-regulated in most of the malignant prostate epithelial cells relative to the normal prostate cells. Among the known effectors of HSF1 action, constitutive levels of HSP70 and HSP90 are not significantly altered by the naturally elevated expression of HSF1 as in PC-3M cells or by transduced overexpression of HSF1 in PC-3 cells. The basal levels of HSP27 in both cases are, however, consistently increased by two- to threefold. With respect to response to heat shock, high basal concentration of HSP90 is not further enhanced in these cells, and HSP70 is up-regulated irrespective of HSF1 level. Heat shock, however, causes an increase in HSP27 when HSF1 is up-regulated, except when the expression of HSF1 is already too high. These results document for the first time that HSF1 is overexpressed in human prostate cancer cells, at least one consequence of which in the prostate cancer cell lines tested is stimulation of both basal and stress-induced expression of HSP27, an important factor in cell growth, differentiation, or apoptosis.

Diverse biological effect and smad signaling of bone morphogenetic protein 7 in prostate tumor cells

We found that bone morphogenetic protein (BMP) 7, a member of the BMP family, was strikingly up-regulated during the development of primary prostatic adenocarcinoma in the conditional Pten deletion mouse model. To determine the relevance of this finding to human prostate cancer, we examined the expression of BMPs and BMP receptors (BMPR) as well as the responsiveness to recombinant human BMP7 in a series of human prostate tumor cell lines. All prostatic cell lines tested expressed variable levels of BMP2, BMP4, and BMP7 and at least two of each type I and II BMPRs. In all cases, BMP7 induced Smad phosphorylation in a dose-dependent manner, with Smad5 activation clearly demonstrable. However, the biological responses to BMP7 were cell type specific. BPH-1, a cell line representing benign prostatic epithelial hyperplasia, was growth arrested at G1. In the bone metastasis-derived PC-3 prostate cancer cells, BMP7 induced epithelial-mesenchymal transdifferentiation with classic changes in morphology, motility, invasiveness, and molecular markers. Finally, BMP7 inhibited serum starvation-induced apoptosis in the LNCaP prostate cancer cell line and more remarkably in its bone metastatic variant C4-2B line. Each of the cell lines influenced by BMP7 was also responsive to BMP2 in a corresponding manner. The antiapoptotic activity of BMP7 in the LNCaP and C4-2B cell lines was not associated with a significant alteration in the levels of the proapoptotic protein Bax or the antiapoptotic proteins Bcl-2, Bcl-xl, and X-linked inhibitor of apoptosis. However, in C4-2B cells but not in LNCaP cells, a starvation-induced decrease in the level of survivin was counteracted by BMP7. Taken together, these findings suggest that BMPs are able to modulate the biological behavior of prostate tumor cells in diverse and cell type-specific manner and point to certain mechanisms by which these secreted signaling molecules may contribute to prostate cancer growth and metastasis.

EphB4 Expression and Biological Significance in Prostate Cancer

Prostate cancer is the most common cancer in men. Advanced prostate cancer spreading beyond the gland is incurable. Identifying factors that regulate the spread of tumor into the regional nodes and distant sites would guide the development of novel diagnostic, prognostic, and therapeutic targets. The aim of our study was to examine the expression and biological role of EphB4 in prostate cancer. EphB4 mRNA is expressed in 64 of 72 (89%) prostate tumor tissues assessed. EphB4 protein expression is found in the majority (41 of 62, 66%) of tumors, and 3 of 20 (15%) normal prostate tissues. Little or no expression was observed in benign prostate epithelial cell line, but EphB4 was expressed in all prostate cancer cell lines to varying degrees. EphB4 protein levels are high in the PC3 prostate cancer cell line and several folds higher in a metastatic clone of PC3 (PC3M) where overexpression was accompanied by EphB4 gene amplification. EphB4 expression is induced by loss of PTEN, p53, and induced by epidermal growth factor/epidermal growth factor receptor and insulin-like growth factor-I/insulin-like growth factor-IR. Knockdown of the EphB4 protein using EphB4 short interfering RNA or antisense oligodeoxynucleotide significantly inhibits cell growth/viability, migration, and invasion, and induces apoptosis in prostate cancer cell lines. Antisense oligodeoxynucleotide targeting EphB4 in vivo showed antitumor activity in murine human tumor xenograft model. These data show a role for EphB4 in prostate cancer and provide a rationale to study EphB4 for diagnostic, prognostic, and therapeutic applications.

Increased expression of osteopontin contributes to the progression of prostate cancer

Osteopontin is a secreted glycosylated phosphoprotein known to be involved in numerous physiologic functions and associated with the late stages of various cancers. We used preneoplastic and neoplastic mouse models of prostate cancer to determine the onset of elevated expression of osteopontin in the development of this disease. Osteopontin alterations occurred early in the disease with dysregulated expression observed in lesions of low-grade prostatic intraepithelial neoplasia (PIN). Over time, osteopontin expressing dysplastic cells seemed to increase in number in high-grade PIN and increased further in adenocarcinoma, and in metastasis, almost all of the cancer cells immunohistochemically stained positive for osteopontin overexpression. We examined the biological properties of human prostate cancer cell lines LNCaP and PC-3, in which osteopontin overexpression was achieved via lentiviral gene transduction. Evidence was obtained that osteopontin could contribute to a proliferative advantage in both cell types, although more significantly in LNCaP than PC-3. Osteopontin also influenced their in vitro invasive ability, and again, most strikingly in the weakly oncogenic LNCaP. Furthermore, excess osteopontin induced the LNCaP cells to acquire a strong intravasation potential in vivo in the chicken embryo chorioallantoic membrane assay for blood vessel penetration. These results establish a correlation between an increased gradient of osteopontin expression throughout the stages of murine prostate cancer, beginning from the preneoplastic lesions to distant metastases that suggests a proliferative and invasive advantages to those prostate tumor cells overexpressing osteopontin. Together, these findings support a strategy designed to target osteopontin in the context of prostate cancer therapy.

Inactivation of Apc in the Mouse Prostate Causes Prostate Carcinoma

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Cancer-associated fibroblasts enhance the gland-forming capability of prostate cancer stem cells.

Signals originating from cancer-associated fibroblasts (CAF) may positively regulate proliferation and tumorigenicity in prostate cancer. In this study, we investigated whether CAFs may regulate the biology of prostate cancer stem cells (CSC) by using a conditional Pten deletion mouse model of prostate adenocarcinoma to isolate both CAF cultures and CSC-enriched cell fractions from the tumors. CSCs that were isolated possessed self-renewal, spheroid-forming, and multipotential differentiation activities in tissue culture, segregating with a cell fraction exhibiting a signature expression phenotype, including SCA-1 (high), CD49f (high), CK5 (high), p63 (high), Survivin (high), RUNX2 (high), CD44 (low), CD133 (low), CK18 (low), and Androgen Receptor (low). CSC spheroid-forming efficiency was differentially influenced by the nature of fibroblasts in a coculture system: Compared with mouse urogenital sinus mesenchyme or normal prostate fibroblasts, CAFs enhanced spheroid formation, with the spheroids displaying generally larger sizes and more complex histology. Graft experiments showed that CSCs admixed with CAFs produced prostatic glandular structures with more numerous lesions, high proliferative index, and tumor-like histopathologies, compared with those formed in the presence of normal prostate fibroblasts. Together, our findings underscore a significant role of CAFs in CSC biology.